This invention relates to methods for recovering, in a purified and biologically active form, proteins which are initially produced as insoluble, biologically inactive inclusion bodies in microorganisms that have been transformed with recombinant DNA expression vectors which direct expression of the proteins. More particularly, the invention relates to methods of recovering such proteins in improved yields by minimizing losses which occur during processing as a result of protein aggregation and precipitation.
Recombinant DNA technology allows the insertion of a vector carrying foreign ("heterologous") DNA into a microorganism in a manner which allows the heterologous DNA to be expressed; that is, the vector contains genetic instructions which direct the microorganism to produce a protein which is encoded by a portion of the heterologous DNA sequence. By growing transformant microorganisms in a fermentor and subjecting them to conditions under which the heterologous DNA is expressed, valuable proteins can be produced in large quantity at relatively low cost.
Unfortunately, many heterologous proteins which are produced in transformant microorganisms do not fold into their native three-dimensional conformation in the host cell environment. Improper folding of the expressed protein has several untoward consequences. In the first place, the improperly folded proteins tend to form aggregates which are insoluble within the host cell. These insoluble aggregates are recognizable within the cell as "inclusion bodies", sometimes also referred to as "refractile bodies." The formation of inclusion bodies may also be partially caused by oligomerization of the protein through the formation of intermolecular disulfide bonds. Not only are the improperly folded proteins insoluble, but also they are biologically inactive. As exemplary of heterologous proteins which form insoluble, biologically inactive inclusion bodies upon expression in a host cell, one can mention animal growth hormones such as bovine growth hormone and porcine growth hormone.
In order to produce useful proteins, it is necessary to convert the improperly folded inclusion body proteins into their native conformations, in which they are soluble and biologically active. Moreover, it is necessary to purify the proteins in order to remove contaminating cell debris and host cell proteins. A number of schemes have been proposed for converting inclusion body proteins into their soluble, native conformations and for purifying the proteins to produce commercially acceptable products. All of the proposed schemes are characterized by an initial unfolding or denaturing step in which the inclusion body proteins are treated with a strong denaturing agent (sometimes referred to as a chaotrope) in order to unfold the protein molecules and render them soluble. Guanidine hydrochloride is the most commonly employed strong denaturant for this purpose. At a subsequent stage in the recovery process, the denaturing agent is removed so that the unfolded protein molecules can refold into their native conformation, a process also referred to herein as "renaturation".
U.S. Pat. No. 4,511,503 discloses a typical recovery scheme of the type just described. A number of variations on this scheme, including additional processing steps directed to purification and/or yield enhancement, have been proposed. Thus, for example, U.S. Pat. No. 4,511,502 discloses a process wherein the solubilized protein/denaturant solution is passed over a molecular sieve or centrifuged at high speed to remove higher molecular weight components. U.S. Pat. No. 4,518,526 discloses a process in which a transformant cell culture is treated with a buffered solution having sufficient ionic strength to solubilize most of the host cell protein while the heterologous protein remains insoluble. The cells are then lysed, the supernatant containing the solubilized host cell protein removed and the insoluble inclusion bodies solubilized in the strong denaturant.
Other publications disclosing denaturation/renaturation schemes for converting inclusion body proteins into their soluble, native conformations include PCT publication No. WO 83/04418, European Patent Application Publication No. 0 123 928, European Patent Application Publication No. 0 121 775, European Patent Application Publication No. 0 116 778 and European Patent Application Publication No. 0 114 507.
At some point in the recovery process, it is necessary to subject the solubilized protein to a purification step in order to remove contaminants such as unwanted host cell proteins. Conventional techniques of protein purification including ion-exchange chromatography, affinity chromatography and the like are generally employed for this purpose. The purification step is performed, in some cases, prior to removal of the denaturing agent and, in other cases, subsequent to the removal of the denaturing agent. Theoretically, the denaturation/renaturation schemes discussed above provide a facile solution to the problem of recovering inclusion body proteins in soluble, biologically active form free of contaminants. The practical implementation of these schemes, however, has been plagued by problems of low yield and uneconomical operation. These problems result largely from the tendency of the solubilized protein to reaggregate, either because the proteins refold improperly upon removal of the denaturant or because the conditions under which purification is carried out interfere with the ability to maintain the protein in a soluble form. We have found that the use of guanidine solubilization followed by removal of the guanidine to refold the protein and purification on an ion-exchange chromatography column results in product recoveries from about 4% to about 12%, based on the amount of desired protein which is present in the inclusion bodies. These yields are far below those which are considered minimally acceptable from a commercial standpoint.